UBC Theses and Dissertations
The 503nm pigment of Escherichia coli: properties and function Kamitakahara-Pearlstone, Joyce Reiko
The streptomycin (Sm)-dependent mutants of four Escherichia coli strains (known to be catabolite-repression negative) were found to have a 25-35% lower aerobic efficiency (yield of protein from glucose) than the parent wild-type organisms. A non-dependent revertant derived from the Sm-dependent mutant of JE. coli B was also lower in efficiency. In contrast, the anaerobic protein yields (although lower than that from aerobic growth) were identical for both types of cells. In both wild-type and mutant strains the glucose-reduced/peroxide-oxidized difference spectra of whole cells showed the same content of cytochromes and flavin. However, Sm-dependent strains lacked the 503nm pigment which was present in all wild-type strains. These observations suggested that the 503nm pigment (P503) (of previously unknown function) might play a role in energy metabolism. Addition of gluconate to air-oxidized cells produced the P503 peak prior to the appearance of cytochrome and flavin absorption bands. Addition of succinate, glycerol, lactate or acetate produced cytochrome and flavin spectra but not P503. Addition of the amino acid L-methionine to air-oxidized cells elicited the P503 band rapidly but the other components of the difference spectrum did not appear until later. No other amino acid tested had this specific effect on P503. When wild-type cells were grown on limiting glucose-salts medium containing 2,4-dinitrophenol (500 µM) , the yield of cell protein was decreased and formation of P503 was inhibited. Also, growth under these conditions resulted in derepressed levels of fumarase, aconitase and, unexpectedly, glucose 6-phosphate dehydrogenase. From these observations the general hypothesis was developed that P503 participates in an oxidative energy-yielding pathway in which the initial substrate is reduced nicotinamide adenine triphosphate (NADPH) , (the first product of gluconate metabolism in glucose-grown E. coli). The synthesis of P503 was observed with glucose or gluconate as carbon source. Less P503 was synthesized when succinate, glycerol or lactate was the carbon source, in which cases generation of NADPH would be less efficient. When L-methionine was present in medium containing glucose as carbon source, the synthesis of P503 was inhibited. Other amino acids did not inhibit synthesis of P503. The unique response of P503 to methionine suggests regulation of the pigment by this amino acid. In agreement with observations of other investigators, P503 was found to be transient and labile, and could not be detected in cells which had been frozen and thawed or in cell extracts.
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